Reverse cholesterol transport (RCT) is the major mechanism by which cholesterol is transported from the peripheral tissues including macrophages associated with the artery wall, to liver for the ultimate conversion into bile acids or direct secretion into the bile. Within the cells or while being transported in the blood stream associated with the lipoproteins, cholesterol is mainly present as cholesteryl esters (CE). Thus, the obligatory first and rate-limiting step of RCT is the intracellular hydrolysis of the stored intracellular CE in the peripheral tissues, e.g., macrophage foam cells, and this reaction is catalyzed by a neutral cholesteryl ester hydrolase (CEH). Free or unesterified cholesterol (FC) that is removed by extra-cellular cholesterol acceptors such as HDL is re-esterified by serum LCAT and carried as CE to the liver where it is delivered by selective uptake via scavenger receptor BI (SR-BI). Hydrolysis of CE once again is obligatory to subsequent conversion of released FC to bile acids or direct secretion into bile. Having established the role of human macrophage CEH in regulating the efflux of FC, RCT and thus attenuating diet-induced atherosclerosis in LDLR-/- mice, the PI has recently reported the cloning and characterization of human liver CEH. Over-expression of this enzyme results in intracellular CE mobilization and an increase in bile acid synthesis. Adenovirus- mediated over-expression of CEH in mice led to significant increase in in vivo RCT and increased elimination of cholesterol as secreted bile acids, and this process required the presence of HDL- receptor SR-BI. CEH-mediated CE hydrolysis, therefore, represents a key event regulating the first step in RCT (generation of free cholesterol in macrophage for efflux) as well as the final step (generating free cholesterol for bile acid synthesis or biliary cholesterol secretion). The central hypothesis of this research project is: Hepatic CEH affects RCT by regulating the hydrolysis of intracellular cholesterol esters (endogenously synthesized or delivered via selective uptake from HDL through SR-BI) thereby providing free cholesterol for elimination as bile acids or direct secretion into the bile and is, therefore, potentially anti-atherosclerotic. This hypothesis will be tested by the following four specific aims: Aim 1: To establish the anti-atherosclerotic role of hepatic CEH by developing liver specific CEH transgenic mice. Aim 2: To delineate the mechanism(s) underlying hepatic CEH-mediated regulation of RCT: role of CEH in hydrolyzing CE delivered to liver by scavenger receptor BI (SR-BI) or SR-BII mediated uptake of HDL-associated CE. Aim 3: To determine the role of hepatic CEH in regulating FC availability for neutral or acidic pathways for bile acid synthesis. Aim 4: To obtain in vivo proof of concept by liver-specific targeted disruption of CEH in mice and to determine its effect on intracellular CE metabolism and atherosclerosis. PUBLIC HEALTH RELEVANCE: Liver is the only organ responsible for the ultimate elimination of cholesterol from the body as free cholesterol (FC) or bile acids that are excreted in the feces. Cholesterol associated with excess unmodified LDL as well as the cholesterol removed from the peripheral organs including artery wall- associated macrophage foam cells by HDL is taken up by the liver. Both these lipoproteins deliver cholesterol in the form of cholesterol esters (CE) and within liver hydrolysis of these CEs is essential to provide FC for bile acid synthesis or for direct elimination into the bile. This hydrolysis is carried out in the extra-lysosomal compartment by a neutral cholesteryl ester hydrolase (CEH). We have identified the human hepatic CEH and demonstrated its role in increasing bile acid secretion as well as in enhancing the flux of cholesterol from macrophages to bile suggesting an anti-atherosclerotic function of hepatic CEH. The proposed studies will build on this foundation and establish the role of hepatic CEH in not only increasing cholesterol elimination from the body as bile acids but also in attenuating diet-induced atherosclerosis. Given the prevalence of atherosclerosis and coronary artery disease, the current findings are likely to have important clinical relevance.